Microsatellite Expansion Diseases
Aberrant expansion of microsatellites in DNA is associated with a number of neurological and neuromuscular diseases (O'Donnell, W T, Warren, S T (2002), Annu. Rev. Neurosci. 25: 315). These diseases are caused by microsatellite repeat expansions in coding and non-coding regions. The characterized coding region expansion diseases include Dentatorubral pallidoluysian atrophy (DRPLA), Huntington chorea (HD), Oculopharyngeal muscular dystrophy (OPMD), Spinobulbar muscular atrophy (SBMA), and Spinocerebellar ataxia types 1, 2, 3, 6, 7, and 17 (SCA1, SCA2, SCA3, SCA6, SCA7, SCA17). The characterized non-coding region expansion diseases include Fragile XA, Fragile XE, Friedrich's ataxia, Myotonic Dystrophy type 1 (DM1), Myotonic Dystrophy type 2 (DM2), and Spinocerebellar ataxia types 8, 10, and 12 (SCA8, SCA10, SCA12). Huntington's disease-like type 2 (HDL2) is likewise caused by a microsatellite expansion.
Microsatellite expansion diseases have been most commonly associated with trinucleotide expansion mutations. In fact, at least 16 of the microsatellite expansion diseases reported to date have been characterized as trinucleotide expansion diseases. More recently, however, microsatellite expansion diseases have also been associated with tetranucleotide and even pentanucleotide expansion mutations. Disease severity and age of onset have both been related to the size of the expansion mutation, eventually leading to muscle weakness and premature cataract formation, and, in severe cases, to hypotonia, muscle heart block, and nervous system dysfunction (Korade-Mirnics, Z, Babitzke, P, Hoffman, E (1998) Nuc. Acids Res. 26(6): 1363-1368).
Myotonic dystrophy (dystrophia myotonica, DM) is a multisystemic, dominantly inherited disorder often characterized by myotonia, or, delayed muscle relaxation due to repetitive action potentials in myofibers, and muscle degeneration. Manifestations of DM may also include heart block, ocular cataracts, hypogonadism, and nervous system dysfunction.
Myotonic dystrophy type 1 (DM1) is caused by a trinucleotide (CTG)n expansion (n=50 to >3000) in the 3′-untranslated region (3′UTR) of the Dystrophia myotonica-protein kinase (DMPK) gene. Myotonic dystrophy type 2 (DM2) is caused by a tetranucleotide (CCTG)n expansion (n=75 to ˜11,000) in the first intron of zinc finger protein 9 (ZNF9) gene (Ranum, L P W, Day, J W (2002) Curr. Opin. in Genet. and Dev. 12:266-271).
Although the expansions are located on different chromosomes, there appears to be a common pathogenic mechanism involving the accumulation of transcripts into discrete nuclear RNA foci containing long tracts of CUG or CCUG repeats expressed from the expanded allele (Liquori C L, Ricker K, Moseley M L, Jacobsen J F, Kress W, Naylor S L, Day J W, Ranum L P (2001), Science 293: 864-867).
In effect, both DM1 and DM2 mutant transcripts accumulate as foci within muscle nuclei (Liquori, et al., 2001). An indication that these transcripts are pathogenic comes from studies on HSALR mice, which express a large CTG repeat in the 3′-UTR of a human skeletal actin transgene (Mankodi, A, Logigian, E, Callahan, L, McClain, C, White, R, Henderson, D, Krym, M, Thornton, C A (2000) Science 289: 1769-1773). These transgenic mice develop myonuclear RNA foci, myotonia, and degenerative muscle changes similar to those seen in human DM. The myotonia in HSALR mice is caused by loss of skeletal muscle chloride (ClC-1) channels due to aberrant pre-mRNA splicing (Mankodi, A, Takahashi, M P, Jiang, H, Beck, C L, Bowers, W J, Moxley, R T, Cannon, S C, Thornton, C A (2002) Mol. Cell 10: 35-44). Similar ClC-1 splicing defects exist in DM1 and DM2. However, the connection between accumulation of mutant DM transcripts in the nucleus and altered splice site selection has not been established (Faustino, N A, Cooper, T A (2003) Genes Dev. 17: 419-437).
The RNA gain-of-function hypothesis proposes that mutant DM transcripts alter the function and localization of alternative splicing regulators, which are critical for normal RNA processing. Consistent with this proposal, misregulated alternative splicing in DM1 has been demonstrated for six pre-mRNAs: cardiac troponin T (cTNT), insulin receptor (IR), muscle-specific chloride channel (ClC-1), tau, myotubularin-related protein 1 (MTMR1) and fast skeletal troponin T (TNNT3) (Kanadia R N, Johnstone K A, Mankodi A, Lungu C, Thornton C A, Esson D, Timmers A M, Hauswirth W W, Swanson M S (2003), Science 302: 1978-1980).
In all cases, normal mRNA splice variants are produced, but the normal developmental splicing pattern is disrupted, resulting in expression of fetal protein isoforms that are inappropriate for adult tissues. The insulin resistance and myotonia observed in DM1 correlate with the disruption of splicing of two pre-mRNA targets, IR and ClC-1, respectively (Savkur R S, Philips A V, Cooper T A, Dalton J C, Moseley M L, Ranum L P, Day J W (2004), Am J Hum Genet 74:1309-1313).
The mechanism by which expanded repeats alter the regulation of pre-mRNA alternative splicing is unclear. Two families of RNA-binding proteins have been implicated in DM1 pathogenesis: CUG-BP1 and ETR-3-like factors (CELF) and muscleblind-like (MBNL) proteins (Ladd A N, Charlet-B N, Cooper T A (2001), Mol Cell Biol 21: 1285-1296). Six CELF (also called BRUNOL) genes have been identified in humans (Ladd A N, Nguyen N H, Malhotra K, Cooper T A (2004), J Biol Chem 279: 17756-17764). All six CELF proteins have been shown to regulate pre-mRNA alternative splicing and two (CUG-BP1 and ETR-3/CUG-BP2) have been shown to have cytoplasmic RNA-associated functions (Mukhopadhyay D, Houchen C W, Kennedy S, Dieckgraefe B K, Anant S (2003), Mol Cell 11: 113-126).
A functional link has been established between splicing regulation by CELF proteins and DM1 pathogenesis. CUG-BP1 regulates alternative splicing of at least three of the pre-mRNAs (cTNT, IR and ClC-1) that are misregulated in DM striated muscle (Charlet-B N, Savkur R S, Singh G, Philips A V, Grice E A, Cooper T A (2002b), Mol Cell 10: 45-53). The splicing patterns observed for all three pre-mRNAs are consistent with increased CUG-BP1 activity and an increase in CUG-BP1 steady-state levels in DM1 striated muscle (Charlet-B N, Savkur R S, Singh G, Philips A V, Grice E A, Cooper T A (2002b), Mol Cell 10: 45-53).
Furthermore, cTNT minigenes expressed in DM1 muscle cultures or cTNT and IR pre-mRNAs co-expressed with CUG repeat RNA in normal cells reproduce the aberrant splicing patterns observed for endogenous genes in DM cells (Philips A V, Timchenko L T, Cooper T A (1998), Science 280: 737-741; Savkur R S, Philips A V, Cooper T A (2001), Nat Genet 29: 40-47). The trans-dominant effects of endogenous or co-expressed CUG repeat RNA on cTNT and IR splicing regulation require the intronic CUG-BP1-binding sites, indicating that binding by CUG-BP1 and/or other CELF family members to their cognate intronic regulatory elements is required for induction of aberrant splicing regulation by CUG repeat RNA (Philips A V, Timchenko L T, Cooper T A (1998), Science 280: 737-741; Savkur R S, Philips A V, Cooper T A (2001), Nat Genet 29: 40-47).
The CNS symptoms of DM1 may include cognitive impairment, hypersomnolence, heightened sensitivity to anesthetic agents, central hypoventilation, neuroendocrine dysfunction, and effects on personality and behavior [reviewed by Harper (Harper, P. S. (2001), Myotonic dystrophy. Saunders London) and Ashizawa (Ashizawa, T. (1998), Arch. Neurol., 55, 291-293)]. Some of these effects, such as, mental retardation in individuals with congenital DM1, occur during development (Dyken, P. R., Harper, P. S. (1973), Neurology, 23, 465-473). Other symptoms, such as, hypersomnolence, appear during adult life. The mechanism and neuropathologic correlates for CNS involvement in DM1 are unknown.
It is presently unclear whether any steps in the pathogenic sequence of poly(CUG) expression, formation of RNA inclusions, sequestration of RNA binding proteins, and disruption of alternative splicing can take place in the CNS. There is controversy about which cells in the mature brain, if any, express DMPK (Lam, L. T., Pham, Y. C., Nguyen, T. M., and Morris, G. E. (2000), Hum. Mol. Genet., 9, 2167-2173).
Microtubule-associated protein tau (MAPT) pre-mRNA is alternatively spliced at exons 2, 3, and 10 (Goedert, M., Spillantini, M. G., Jakes, R., Rutherford, D., and Crowther, R. A. (1989), Neuron, 3, 519-526). Tau transcripts in fetal brain do not include exon 10, whereas ˜50% of transcripts in adult brain include this exon which encodes an additional microtubule binding domain (Hong, M., Zhukareva, V., Vogelsberg-Ragaglia, V., Wszokek, Z., Reed, L., Miller, B. I., Geschwind, D. H., Bird, T. D., McKeel, D., Goate, A. et al. (1998), Science, 282, 1914-1917). Alternative splicing of exons 2 and 3 also is developmentally regulated (neither exon is included in the fetus, adults mainly include exon 2).
The relative proportion of tau splice products is tightly regulated, as shown by kindreds with frontotemporal dementia and parkinsonism (FTDP-17) due to mutations in MAPT. Silent mutations in MAPT exon 10, or, in the flanking intron, lead to FTDP-17 by disrupting cis elements that regulate splicing of tau pre-mRNA (D'Souza, I., Poorkaj, P., Hong, M., Nochlin, D., Lee, V. M., Bird, T. D., and Schellenberg, G. D. (1999), Proc. Natl. Acad. Sci. U.S.A, 96, 5598-5603). Usually these mutations lead to increased inclusion of exon 10 (Lee, V. M., Goedert, M., and Trojanowski, J. Q. (2001), Annu. Rev. Neurosci., 24, 1121-1159). However, some MAPT mutations that segregate with FTDP-17 have the opposite effect of reducing exon 10 inclusion (Stanford, P. M., Shepherd, C. E., Halliday, G. M., Brooks, W. S., Schofield, P. W., Brodaty, H., Martins, R. N., Kwok, J. B., and Schofield, P. R. (2003), Brain, 126, 814-826).
RNA-binding proteins that regulate alternative splicing bind to sequence-specific elements in the pre-mRNA to enhance or repress inclusion of alternative exons. Aberrant regulation of alternative splicing can cause the expression of inappropriate splicing patterns leading to human disease (Faustino and Cooper, 2003). Myotonic dystrophy constitutes an example of a disease that alters the function of RNA-binding proteins to cause misregulated alternative splicing.